US3596652A

US3596652A - Fluid separatory device

Info

Publication number: US3596652A
Application number: US767374A
Authority: US
Inventors: James W Winkelman
Original assignee: Bio Science Laboratories
Current assignee: Bio Science Laboratories
Priority date: 1968-10-14
Filing date: 1968-10-14
Publication date: 1971-08-03
Anticipated expiration: 1988-08-03

Abstract

The device includes a barrel and plunger assembly for drawing the fluid into a separating chamber, chemical separating means such as an agglutinating agent for separating the phases in the chamber and means in the plunger assembly including a one-way valve for mechanical separation of the less dense supernatant phase from the more dense phase. The device is of particular utility in separation of plasma from whole blood in preparation for chemical analyses of the plasma.

Description

United States Patent 82888 lye- 11 H2DHP n 8 0088 0 2 222 m l lll h 0 R r "n u a n n n n u v. mm m m m m d 2 .H n m m H m madam C uy t -.l monmm m new TU hv B o cfl m BPRSS an a 67792 d 16666 m 99999 U lllll /////.m.m 35099 I 1 1 34629 7 4 3 N. W 42842 am 72465 m nn 3333 PA u m m n W mm .m 8 .u w C W 7 y 1w 5 WC M w amiw w N am m 0 6 JS7OABV f de m s mm B l AFPA .l. 11].] 2 253 7 2247 it [ill [54] FLUID SEPARATORY DEVICE 5 Claims, 4 Drawing Figs.

ABSTRACT: The device includes a barrel and plunger as- 20 NH 25 b 1 6 A l o H k F m 5 8 7 2 P 8 1i 2 M assembly including a one-way valve for mechanical separation of the less dense supernatant phase from the more dense [56] References Cited UNITED STATES PATENTS 657,440 9/1900 McCaw phase. The device is of particular utility in separation of plasma from whole blood in preparation for chemical analyses of the plasma.

Pmcmem 3m:

SHEET 1 BF 2 INVENTOR. James W Wmke/man flTTORNEKS FLUID SEPARATORY DEVICE BACKGROUND OF THE INVENTION 1. Field of the Invention The invention is directed to the separation of liquid phases from fluid mixtures. The invention is more particularly directed to separation of particular liquid phases from fluid mixtures for performing analytical chemical operations on the liquid phases, and in a preferred embodiment, for separating plasma from blood. The term fluid mixture" herein employed to designate liquid compositions of a more or less homogeneous character but which are separable into two or more separate phases of different densities so that such separation in a gravitational field results ultimately in the disposition of the phases as layers, the more dense fluid phases being the lower layers and the less dense fluid phases forming the upper layers. The term fluid as employed with respect to mixtures and phases is intended to refer to solutions, liquid emulsions, colloidal dispersions, suspensions, slurries, suspensions of cells such as blood cells, tissue homogenates or bacterial cultures and the like.

2. Description of the Prior Art The separation of particular liquid phases from fluid mixtures has been carried out by a variety of procedures, one of the most widely used procedures involving centrifugation. In the separation of plasma from blood, for example, a blood sample is drawn from a subject, mixed with an anticoagulant and placed in a centrifuge tube. The tube is placed in the centrifuge which is counterbalanced for the weight of the sample and the sample is centrifuged until the blood cells are sedimented, leaving the plasma as a supernatant fluid. The plasma can then be decanted or pipetted into a suitable container for storage, shipping or immediate clinical chemical analysis. The supernatant plasma is miscible with the more dense, cell-containing phase. The mechanical separation of the plasma requires a high degree of caution on the part of a trained analyst to avoid turbulence in the mixture resulting in resuspension of cells in the plasma. Frequently it may be undesirable to expose the fluid to the atmosphere by transferring it from initial container, to centrifuge tube, to final container as fluids such as blood can contain viruses or micro-organisms which can create a biological hazard to the analyst. Likewise, the fluid to be separated can be contaminated during the separation procedures so that certain analytical results can be affected. Moreover, the separation requires additional equipment such as the centrifuge, the additional tubes, etc. and decantation or pipetting of the supernatant fluid may require a skilled technician to prevent remixing of the separated phases during the mechanical separation thereof. Moreover, in circumstances in which control of temperature is desired during the separation, the operation is complicated by the need to either heat or cool the centrifuge, pipettes, tubes and other auxiliary equipment.

Their is a need for a single simple device for separation of fluids in which separation of fluid mixtures into phases and mechanical separation of phases can be accomplished accurately in the same device while minimizing turbulence and risks of remixing the phases.

SUMMARY OF THE INVENTION The present invention is concerned with apparatus useful for the separation of different fluid phases from a fluid mixture. The invention is particularly directed to apparatus useful of the separation of fluid mixtures into phases of differing densities and the mechanical separation of the phases from each other.

It is an object of the invention to provide a fluid-separatory device in which collection of fluid, phase separation and mechanical separation of phases can be carried out in the same apparatus in a simple procedure involving a minimal number of distinct operations. It is a further object of the invention to provide a fluid-separatory device which is inexpensive and simple in operation and construction, and which can be employed as a container for shipping or storing the fluid phases separated therein. It is a further object of the invention to provide a'device for the separation of a fluid phase such as plasma from whole blood which is adapted to permit the entire operation to be carried out in a single vessel with minimum possibility of cross-contamination between a technician using the device and the sample of blood and a minimum of crosscontamination between phases resulting from turbulence at the interface. A further object is to provide a separatory device which is adapted to operate under a variety of temperature conditions without requiring elaborate provisions for heating and cooling.

The apparatus includes a barrel having a plunger assembly movably disposed therein, so that movement of the plunger unit varies the volume of a sample chamber. The device includes inlet means for introducing a fluid mixture into the chamber and chemical means in the chamber for facilitating the separation of themixture into a plurality of phases of differing densities. The device also includes means in the plunger unit for mechanically separating the phases by permitting one phase to flow through the plunger unit as the plunger unit is moved to the interface of the two phases and preventing such separated phase from flowing back through the plunger unit toward the interface. The inlet means of the device can be coupled to a sampling element such as a hypodermic needle so that collecting the fluidmixture, separation into phases and mechanical separation of one phase from another are accomplished in the same device. The device can also include spacer means which can be employed in lie of or in combination with the chemical means to facilitate the use of the apparatus in a centrifuge. The device is simple in construction and operation and capable of carrying out complete mechanical separation of fluid mixtures with a minimum of cross-contamination between phases or between the fluid mixture and the atmosphere. It can be employed with or without a centrifuge and is thus easily adapted to operation in a refrigerator or water bath or the like.

BRIEF DESCRIPTION OF THE DRAWINGS Other objects and advantages of he invention will be apparent on consideration of the following description and claims and of the drawings wherein:

FIG. 1 is an isometric view of a fluid-separatory device of the invention with a portion 'ofthe barrel and plunger piston broken away to show the chemical separatory means;

FIG. 2 is a cross-sectional view of the device of FIG. 1 taken along line 2-2 of FIG. 1 and showing the device in one state of use;

FIG. 3 is a cross-sectional view of the device of FIG. 1 taken along line 2-2 and showing the device in another state of use;

FIG. 4 is a cross-sectional view of the device of FIG. 1 taken along line 2-2 and showing the device in a third state of use.

DESCRIPTION OF THE PREFERRED EMBODIMENT Referring more particularly to the drawings, the separating apparatus 10 comprises a barrel 12 having side and end walls l4, 16; a plunger member 20, including a plunger piston 22 slidably disposed in the barrel in fluidtight relation to the sidewall 14 thereof so that the plunger piston 22 and the

walls

14, 16 of the barrel define a fluidtight separating chamber 40 of variable volume; disengageable spacer means 70 for selectively limiting movement of the plunger piston 22 in the barrel at a predetermined-point whereby a predetermined minimum volume of chamber) is defined by the piston 22 and

walls

14, 16; closeable inlet mearts 18 on the barrel 12 for introducing fluid into the chamber 40; chemical means 42 for facilitating the separation ofa fluilmixture into two fluid phases, said means being disposedii'n the chamber 40; means for so disposing the chemical means 42 in chamber 40; and conduit means 24 in the plunger piston 22 for providing unidirectional fluid flow from chamber40 through the plungerpis ton 22.

The term fluidtight as herein employed in reference to the relationship of the plunger piston 22 and the barrel sidewall 14 means and refers to that degree of fit of the plunger piston 22 within the barrel which permits fluid to enter or leave the camber 40 only through inlet means 18 or conduit means 24. Such fit permits fluid to be drawn into chamber 40 through the inlet means 18 against the force of gravity by the suction created by movement of the plunger piston away from the end wall 16 of the barrel 12. The fluidtight relation of the plunger piston and the barrel thus corresponds to the relation of the piston and cylinder of a conventional lift pump or the relation of the plunger and syringe barrel of a conventional hypodermic syringe.

The barrel 12 can have any configuration or size compatible with the slidable disposition of the plunger piston 22 therein. A cylindrical configuration similar to that of a syringe barrel is preferred. The dimensions of the barrel are conveniently such as to provide a sample chamber 40 with a maximum volume of from about 5 to about 50 to 100 cubic centimeters. A barrel corresponding to the syringe barrel of a conventional or cubic centimeter hypodermic syringe is convenient. The barrel can likewise be of any suitable material which is sufficiently rigid and fluid resistant to contain the fluids to be separated. Glass, metal, hard rubber or synthetic plastic materials are representative materials. It is greatly preferred that the barrel 12 be transparent, so that movement of the plunger piston 22 therein and separation of fluid phases can be easily observed. Consequently, it is greatly preferred that the barrel be constructed of a transparent material such as glass, or a transparent plastic such as polyethylene, polypropylene or polystyrene.

The closeable inlet means 18 is preferably disposed in the end wall 16 of the barrel 12 and comprises an opening or port through the end wall 16 to provide communication between the inside and outside of the barrel. In a preferred embodiment, the inlet means 18 also includes a tubular nipple 19. The nipple 19 is adapted to be coupled to a fluid-sampling device 36 by friction fit of an adapter 38 about the nipple. Sampling device 36 can be a conventional hollow hypodermic needle. Alternately, the adapter 38 and sampling device or needle 36 can be removed and a fluidtight cap 32 can be releaseably secured over the nipple 19 by friction fit as shown in FIG. 3, thus selectively closing the inlet means. The inlet means must be capable of closure to prevent flow of fluid from sample chamber 40 at certain times during the operation of the device, although the means by which closure is accomplished are not critical. Thus, other means may be employed to provide for selectively closing the inlet means 18. For example, a resilient plug can be inserted into the inlet means, or the nipple can be coupled to a sampling device by means ofa resilient tubular adapter which can be closed by a clamp or the like. Also, the inlet means 18 can have a one-way valve permitting fluid to enter chamber 40 through the inlet means 18, but preventing the exit of fluid through the inlet means.

The plunger unit 20 and piston 22 can have any configuration which corresponds to the slidable fluidtight disposition of the plunger piston 22 in the barrel 12. The plunger unit 20 can also include a plunger rod 21 which extends upwardly from the piston 22 and away from the end wall 16 of the barrel. Preferably, the plunger piston 22 and plunger rod 21 are formed as a unit from the same type of material employed in the manufacture of the barrel. The combined length of both plunger elements should be such that a portion of the plunger rod 21 extends beyond the sidewall 14 of the barrel 12 when the plunger piston 22 is disposed against the end wall 16 of the barrel. Both the barrel l2 and the plunger rod 21 can have manually graspable protrusions 60 extending outwardly therefrom to facilitate manual control of the sliding motion of the plunger piston 22 in the barrel 12.

The apparatus 10 can also include a disengageable spacer 70 adapted to fit between a protrusion 60 on the barrel and a corresponding protrusion 60 on the plunger rod 21 as shown in FIG. 2. The spacer 70 can be of any suitable material of sufficient rigidity to maintain the plunger rod and

piston

21, 22 in a predetermined spaced relation from the end wall 16 of barre] 12 when the apparatus is employed in a centrifuge. When 70 is engaged with protrusions 60 as shown in FIG. 2, the downward motion of plunger piston 22 is limited so that the volume of chamber 40 cannot decrease below a predetermined limit. The dimensions of space 70 are selected so that the predetermined minimum volume of chamber 40 is at least as great as the volume of fluid mixture desired to be separated. When the apparatus and fluid mixture are centrifuged, the spacer 70 prevents the plunger piston 22 from moving under centrifugal force. A second disengageable spacer adapted to provide a second predetermined volume to chamber 40 can also be employed. Such second volume is equal to or preferably slightly greater than the volume of the more dense fluid to be retained in chamber 40 after the less dense supernatant fluid is mechanically removed. A single rectangular plate having a length and breadth corresponding to the two predetermined volumes, respectively, is conveniently employed as a spacer.

The unidirectional conduit means 24 extends through the piston 22 and includes a one-way check vale 2. The exact type of one-way check valve to be employed is not critical and a ball valve, butterfly valve, spring-biased valve or the like can be employed. However, it is essential that the valve 26 and conduit be such as to permit fluid to flow upward through the plunger piston 22 from chamber 40 and to prevent any flow of fluid downward through the piston 22 into chamber 40. A one-way, gravity-biased ball valve gives excellent performance in providing the required unidirectional flow, and a ball valve is the preferred one-way valve. In the valve 26, a ball 25 is disposed in a valve chamber 27 which comprises an enlarged portion of the conduit 24. In the upper portions of the valve chamber 27, a stop 29 extends into chamber 27. Stop 29 is so disposed in the upper portion of chamber 27 as to prevent the ball 25 from blocking fluid flow from chamber 27 upward, and is of such dimensions as to permit upward flow even when the ball is in its uppermost position. When the device 10 is in its normal vertical position in which the barrel end wall 16 is below the plunger piston 22, the ball 25 is biased by gravity into a position blocking the conduit means as shown in FIGS. 1 and- 2. Suction created in chamber 40 as its volume is increased by upward sliding of the plunger piston 22 and increasing the volume of chamber 40 as shown in FIG. 2, or the weight of any fluid disposed in the barrel 12 above the piston 22 as shown in FIG. 4 provides an additional bias toward closure of the ball valve. The upward pressure of any fluid in chamber 40 results in upward and/or lateral displacement of the ball 25, opening valve 26 to permit fluid flow through the conduit means 24 as illustrated in FIG. 3. Downward sliding motion of the plunger piston 22 in the barrel 12 when the input means 18 are closed increases fluid pressure in chamber 40 as the volume of chamber 40 decreases and results in opening the valve 26. Thus, the one-way valve 26 is adapted to permit unidirectional flow from the chamber 40 through the valve when the plunger piston is moved downward or toward end wall 16.

The device 10 preferably includes receptacle means for collecting fluid which passes from chamber 40 through the plunger piston 22. In a preferred embodiment shown in the drawings, the plunger rod 21 extends upwardly from the plunger piston 22 and entirely surrounds the conduit means 24, whereby the plunger piston 22 and rod 21 define a fluid receptacle or chamber 80. Receptacle is thus disposed downstream of he plunger piston 22 and conduit means 24 with respect to unidirectional flow of fluid therethrough. Receptacle or chamber 80 is closed at another end by an end wall 33 which can be a portion of the plunger rod 21. End wall 33 includes a sealable aperture 30 surrounded by a nipple 31 extending outwardly from end-wall 33. A removable sealing cap 34 can be releasably secured over nipple 31 to seal the aperture 30 as shown in FIGS. 1 and 2. With the cap 34 over the nipple, the pressure of air in receptacle 80 resists the flow of fluid through conduit 24 and permits the device 10 to be handled easily without entry of fluid into receptacle 80 until such flow is desired. The cap 34 can be removed when desired to permit air enclosed in fluid receptacle 80 to escape to the atmosphere through aperture 30 as fluid passes through conduit means 24 into receptacle 80. Tl-lus, the aperture 30 and cap 34 provide adjustable means for relief of pressure in the receptacle 80 by permitting selective venting to the atmosphere. The plunger piston 22, plunge rod 21 and receptacle 80 included therein, and the end wall 33 are preferably formed as a single plunger unit which can be separated from the barrel when a fluid phase sample has been collected in receptacle 80. On separation of the plunger unit 20 from barrel 12 after using the device, the device serves as a pair of separate containers for separated fluids. Conventional means can be used for sealing conduit 24 after separation of the plunger unit and barrel such as a plug (not shown) adapted to fit into the conduit by friction fit, screw threads or the like, or a cap (not shown) adapted to fit over the lower end of the plunger assembly in substantially the same manner as cap 34 fits over nipple 31.

The chemical means 42 disposed in chamber 40 can be disposed therein in any fashion which will ensure that the chemical means act on the fluid mixture to be separated and to separate the same into two phases. Thus, the chemical means can be present as a solid or a liquid in chamber 40, for example, as a liquid film or a solid coating on the interior of the barrel sidewall 14 or on the surface of the plunger piston 22 or the barrel end wall 16, in which case the film or coating serves as means for introducing the chemical separating means into a fluid mixture disposed in chamber 40. The film or coating is applied to the piston or interior of the barrel by removing the plunger unit from the barrel, applying the chemical means and replacing the plunger unit in the barrel. Alternately, it can be present as a mixture with thefluid to be separated, having been mixed with such fluid either prior to or subsequent to the placing of the fluid in the device 10, in which case the inlet 18 serves as means for introducing the chemical separating means into chamber 40 and thus into contact with the fluid mixture. It is greatly preferred, however, that the chemical means be disposed in chamber 40, whether or not a fluid to be separated is simultaneously present.

The chemical means can be any agent suitable for enhancing separation of the particular liquid mixture into two separate phases. When the liquid is an aqueous emulsion such as emulsions of various organic liquids and water, or of fats or oils and water, the chemical means can be an emulsion-breaking agent. When it is desired to separate colloidal dispositions or protein suspensions or solutions into supernatant liquid and a liquid phase containing solid material, a flocculant or a protein precipitant can be employed. When the liquid mixture is a suspension of cells such as blood, aqueous bacterial cultures or tissue homogenates, the chemical agent can be agglutinant, that is, an agent which enhances the agglutination or clumping together of cellular material. In particular applications, selective chemical separating agents capable of separating particular liquid mixtures into particular fluid phases can be employed; for example, when the liquid mixture is milk, an emulsion-breaking agent such as amyl alcohol can be employed to separate the fat from the heavier, aqueous colloidal phase or a protein precipitant such as silver nitrate or lead acetate can be employed to separate the milk into a proteinfree liquid phase and a liquid phase containing precipitated flocculated protein. The chemical separating means cooperates with the other elements of the device to facilitate the complete separation of the desired fluid phase and, in many cases, the chemical separating means determines-the nature of the supernatant fluid separated from the fluid mixture. The exact chemical separating means to be employed is dependent upon the particular fluid mixture to be separated by the use of the device and the particular liquid phases to be obtained, it being essential only that the chemical means facilitate the separation of the liquid mixture into two or more separate liquid phases of different specific gravities.

Various chemical separating agents which can be employed in the separation of particular fluid mixtures are disclosed by Clayton, Theory of Emulsions,5th Edition, chapter Xlll, Blakiston Company, New York (I954) and by Becher, Emulsions: Theory and Practice, Second Edition, chapters 5 and 9, ReinholdQNew York (i965). Agents useful in precipitation of colloidal materials or suspensions are disclosed by Shaw, introduction to Colloid and Surface Chemistry, chapters 8 and 10, Butterworths, London (1966). Chemical means useful in the separation of biological fluid phases in fluid mixtures such as blood are disclosed by Henry, Clinical Chemistry: Principles and Techniques, Hoeber Division of Harper and Rowe (Second Printing, 1964). I

The device of theinvention has particular utility in the separation of plasma or serum from whole blood, typically in preparation for chemical analysis of various plasma or serum components. In such use, the chemical separating means 42 comprises an agglutinant and, optionally, an anticoagulant, and preferably both an agglutinant and an anticoagulant. The agglutinant serves to bring about the clamping together and precipitation of the blood cells and to separate the blood into a cell-free liquid phase and a more dense liquid phase containing the precipitated clumps of cells. Representative agglutinants which can be employed as chemical separating means include fibrinogen, polyvinylpyrrolidone, hemagglutinin, phytohemagglutinin and dextran. The preferred agglutinant is fibrinogen. When an agglutinant is the sole chemical-separating means, clotting of blood takes place as well as agglutination. In such a case,'serum is obtained as the less dense liquid phase above the precipitated cells. Separation of serum rather than plasma is desirable in certain situations. In other cases, plasma is desired, and the loss of plasma components such as prothrombin which are involved in clot formation is undesirable. Thus, in order to separate plasma from blood in the device of the invention, the chemical agent must comprise an anticoagulant, that is, a chemical agent which inhibits the clotting of blood, as well as the agglutinant required to separate the blood cells into a separate liquid phase. Representative anticoagulants which can be employed include heparin and synthetic heparinlike compounds; bishydroxycoumarin; oxalate anticoagulants such as potassium oxalate or ammonium oxalate; citrate anticoagulants such as trisodium citrate, tripotassium citrate and mixtures of trisodium citrate, citric acid and dextrose; alkali metal salts of ethylenediaminetetraacetic acid; potassium fluoride and the like. Heparin is the preferred anticoagulant.

Either the anticoagulant or the agglutinant or both can be disposed in the chamber 40 prior to the introduction'of blood thereinto. Preferably, both the anticoagulant and the agglutinant are disposed as a solid layer or deposit on an internal wall of barrel 12 or the plunger piston 22, and disposition of a solid mixture of the two agents on the surface of end wall 16 is preferred. The chemical means 42 for separation of plasma from blood is conveniently disposed in chamber 40 by withdrawing the plunger unit 20 from the barrel, pouring a solution of heparin and flbrinogen into the barrel and allowing the solvent to evaporate, leaving the mixture of anticoagulant and agglutinant as a solid layer on end wall 16.

Tile amount-of chemical separating means to be employed depends in part on the amount of fluid mixture to be separated and on the result to be achieved. THe chemical separating agent should be employed in an amount such that the dilution and admixture of the agent with the particular fluid mixture provides an effective concentration of the agent in the mixture. An effective concentration is sufficient to produce the desired effect, emulsion breaking, precipitation, clot inhibition, agglutination or the like. In plasma separation for example, the chemical means 42 includesesufiicient heparin to provide an anticoagulant concentration of from about 10 to about 20 to about 50 milligrams of heparin per milliliters of blood, and sufficient fibrinogen to provide an agglutinating amount of from about 50 to'about 200 to about 600 milligrams of fibrinogen per 100 milliliters of blood.

In the operation of the device 10, a fluid mixture 44 to be separated is drawn into chamber 40 through the inlet means 18 as the plunger piston 22 is slidably moved away from barrel end wall 16. Inlet means 18 is then closed by cap 32 and the fluid mixture 44 is mixed with the chemical-separating means 42 which has been disposed in chamber 40 prior to introduction of fluid mixture 44. The device 10 is then maintained in a substantially vertical position as shown in FIG. 2 until the fluid mixture 44 separates into a less dense supernatant fluid phase 52, situated in the upper portion, and a more dense fluid phase 54, situated in the lower portion of chamber 40. Alternately, a spacer 70 is engaged between protrusions 60 on the barrel 12 and plunger rod 21 to maintain the minimum volume of chamber 40 at least equal to the volume of fluid mixture 44, and the device 10 is centrifuged by conventional procedures to dispose the more dense phase 54 in that portion of-chamber 40 nearest end wall 16. Depending upon the fluid mixture employed and the type of separation desired, the liquid phases can be separated by centrifugation while the spacer is engaged without employing a chemical-separating means. The cap 34 is then removed from aperture 30 and when a spacer 70 has been employed, the spacer is disengaged or removed. The plunger piston 22 is then slidably moved downward toward barrel end wall 16. As plunger piston 22 moves into the chamber 40, the upper less dense fluid phase 52 passes through unidirectional conduit 24 and valve 26 into the fluid receptacle 80 as shown in FIG. 3. Downward motion of the plunger piston 22 is continued until the plunger piston 22 reaches the interface 50 between the two liquid phases. Alternately, a second spacer is engaged with a protrusion 60, the second spacer being adapted to prevent downward motion of the plunger piston beyond the interface 50, and the plunger piston 22 is moved downward until further downward motion is prevented by the spacer. Since the less dense liquid in receptacle 80 is prevented from returning to chamber 40 by oneway valve 26, mechanical separation of the two

liquid phases

52, 54 is complete at this stage an the plunger unit can be moved upward as shown in FIG. 4. If desired, cap 32 can be removed from the barrel nipple 19 and the more dense fluid 54 can be drained from chamber 40 through the inlet means 18. Also, the plunger unit 20, including the less dense liquid phase 52 disposed in receptacle 80, can be removed and aperture 30 can be capped and conduit 24 closed in any convenient manner to provide a closed receptacle for storing or handling the fluid phase 52. Since the device can be employed without centrifugation, the temperature at which separation is carried out can be controlled easily by placing the device in a refrigerator, incubator, ice bath, water bath or the like.

In the separation of plasma from whole blood, the chemical means 42 employed is preferably a solid film of heparin and fibrinogen, and the mixture of the anticoagulant and the agglutinant is disposed as a film on end wall 16 as shown in FIG. 1. The chemical means is deposited on the end wall as described above, prior to using the device. The device equipped with a needle 36 and adapter 38 on inlet means 18 and a cap 34 on aperture 30 is employed to draw a sample of blood from a subject into chamber 40. The device 10 is inverted, the needle and

adapter

36, 38 are removed and cap 34 is placed on inlet means 18. With the inlet means thus closed, the anticoagulant and agglutinant are mixed with the blood fluid mixture 44 by inverting the apparatus several times. The device is then placed in an upright position as shown in FIGS. 2-4 and held for a short period while the plasma separates from the blood cells as an upper fluid phase 52. Generally, the separation is sufficiently rapid so that plasma occupies the upper 45 to 55 percent of the sample chamber volume after about 30 minutes or the upper 50 to 60 percent about 1 hour. When it is desired to separate serum or plasma from whole blood by employing the device in conjunction with a centrifuge, a spacer 70 is employed as described above. When a centrifuge and spacer are employed in separating plasma, the chemical separating means can comprise an anticoagulant alone. With serum, rather than plasma, is separated in this procedure, the chemical means can be omitted or can comprise an agglutinant alone. After the phases have separated to the desired extent, cap 34 is then removed from aperture 30 and the spacer 70, if used, is disengaged. The plunger piston 22 is then urged downward toward the interface 50 of the plasma phase and the blood cell phase. The plasma flows through the conduit means 24 into receptacle as shown in FIG. 3. If desired, the plunger piston can be moved slowly downward during the holding period to provide for gradual continuous flow of plasma through conduit 24 to receptacle 80 as the separation of phases by agglutination of the cells progresses. When the plunger piston 22 reaches the interface 50 or a point slightly above it, the plunger unit 20 is withdrawn upwardly from the barrel 12 as'shown in FIG. 4. 'Cap 34 is replaced on nipple 31 and, if desired, conduit means 24 can be sealed by a plug or the like. The plasma separated in the device can be shipped or stored in the receptacle 80 and is suitable for analytical clinical chemical purposes.

It will thus be seen that the device can be described as an improved syringe similar to conventional hypodermic syringes. Thus, the device 10 has a barrel l2 equipped with a detachable needle 36 and a plunger unit 20 including a plunger piston 22 and plunger rod 21. In the device of the invention, however, the plunger piston includes conduit means 24 for providing unidirectional flow from the barrel into a receptacle 80 enclosed within the rod 21 and means for selectively preventing loss of fluid from the barrel 12 such as the cap 34 over inlet means 18. Moreover, the device 10 includes chemical separation means 42 in chamber 40 of the barrel 12 and means such as inlet 18 or the removable plunger unit for disposing chemical means 42 in chamber 40. Thus, the device corresponds to a syringe which is adapted to bring about the separation of fluid mixtures disposed .therein by chemically enhanced separation of the fluid mixture into phases and also by mechanical separation of one liquid phase from another.

The device can be modified, for example, by the choice of particular unidirectional flow means in the plunger piston, the choice of particular closeable inlet means on the barrel or means of closing the aperture on the receptacle in the plunger unit. Modification of the' chemical-separating means by the choice of particular means to accomplish particular functions in the separation of particular fluid mixtures can be carried out to adapt the device for use in a variety of separatory applications. Also, it will be apparent that in some. applications, one or more spacers can be employed to permit centrifugation of the device and omission or variation of the chemical separating means. Mechanical means for moving the plunger piston can also be employed, with such means being controlled by timers or sensing means such as photoelectric devices for actuating the mechanical means when a predetermined amount of supernatant fluid has separated from the fluid mixture.

What I claim is:

1. A method for separating a fluid mixture into a plurality of fluid phases of differing densities within a syringe having a barrel, including a sidewall and an end wall, inlet means in a wall of the barrel for admitting a fluid mixture thereto; a plunger slidably disposed in the barrel in fluidtight relation to the sidewall thereof so as to form a chamber of variable volume in the barrel, means for selectively closing the inlet means; conduit means on the plunger for providing unidirectional fluid flow through the plunger from the chamber; and a receptacle on the plunger surrounding the conduit means and downstream thereof, the method comprising:

a. drawing a fluid mixture to be separated through the inlet means and into the chamber,

b. mixing the fluid mixture in the chamber with a chemical means for facilitating the separation of a fluid mixture into a plurality of fluid phases of differing densities,

c. closing the inlet means, and thereafter d. holding the syringe in a vertical position with the plunger above the defined chamber until the fluid mixture separates into a plurality of phases of differing densities,

e. moving the plunger downward into the chamber toward the interface resulting from separation of the mixture and simultaneously venting the receptacle to atmosphere, whereby the uppermost liquid phase flows through the conduit means into the receptacle;

f. terminating the downward motion at a point in said chamber not below the interface;

g. and thereafter withdrawing the plunger, receptacle and the liquid phase therein from the barrel.

2. The method of claim 1 further comprising the step of sealing the receptacle from co mmunication with atmosphere 22;;3?" UNITED STATES PATENT OF'FICE CERTIFICATE OF CORRECTION Patent No. 3.596,652 Dated 3 August 1221 Inventor(s) James W. Winkelman It is certified that error appears in the above-identified patent and that said Letters Patent are hereby corrected as shown below:

In column 2, line 32, change "lie" to lieu Column 5, line 26, delete "liquid in" and insert liquid disxxjsedg. in"-; in line U8, delete "dispositions" and insert dispersions Column 6, line 21, delete "clamping" and insert clumping Signed and sealed this 11th day of April 1972.

(SEAL) Attest:

EDWARD M.FLETCHER JR. Attesting Officer ROBERT GOT'ISCHALK Commissioner of Patents

Claims

1. A method for separating a fluid mixture into a plurality of fluid phases of differing densities within a syringe having a barrel, including a sidewall and an end wall, inlet means in a wall of the barrel for admitting a fluid mixture thereto; a plunger slidably disposed in the barrel in fluidtight relation to the sidewall thereof so as to form a chamber of variable volume in the barrel, means for selectively closing the inlet means; conduit means on the plunger for providing unidirectional fluid flow through the plunger from the chamber; and a receptacle on the plunger surrounding the conduit means and downstream thereof, the method comprising: a. drawing a fluid mixture to be separated through the inlet means and into the chamber, b. mixing the fluid mixture in the chamber with a chemical means for facilitating the separation of a fluid mixture into a plurality of fluid phases of differing densities, c. closing the inlet means, and thereafter d. holding the syringe in a vertical position with the plunger above the defined chamber until the fluid mixture separates into a plurality of phases of differing densities, e. moving the plunger downward into the chamber toward the interface resulting from separation of the mixture and simultaneously venting the receptacle to atmosphere, whereby the uppermost liquid phase flows through the conduit means into the receptacle; f. terminating the downward motion at a point in said chamber not below the interface; g. and thereafter withdrawing the plunger, receptacle and the liquid phase therein from the barrel.

2. The method of claim 1 further comprising the step of sealing the receptacle from communication with atmosphere during the mixing and holding steps.

3. THe method of claim 1 wherein the chemIcal means for facilitating separation comprises an agglutinant.

4. THe method of claim 3 wherein the chemical means for facilitating separation further comprises an anticoagulant.

5. THe method of claim 4 wherein the anticoagulant is heparin and the agglutinant is fibrinogen.